The present invention relates to the detection of HLA-G. The present invention provides antigens for generating specific antibodies to both soluble and membrane bound HLA-G, as well as exemplary antibodies.
A central question in pregnancy is how the fetal-placental unit avoids maternal immune rejection. Although fetal and maternal cells interact throughout pregnancy, the fetus typically remains a privileged site, not subject to rejection. It is likely that the particular nature of the cells at the fetal-maternal interface and their products help prevent rejection of the fetus by the maternal immune system.
Implantation and placental development physically connect the mammalian embryo to the maternal uterus. Establishing this connection is essential for subsequent development. The initial developmental events which occur in the embryo set aside unique extraembryonic cellular lineages which are the precursors of the placenta. The first differentiation event gives rise to trophoblasts, which are specialized epithelial cells of the placenta that physically connect the embryo and the uterus, see for example Cross et al. (1994), Science 266: 1508 for a review of the events surrounding implantation and formation of the placenta.
After fertilization in the oviduct, a series of cell divisions create a mass of totipotent cells called the morula. The first differentiation event occurs after compaction of the morula, leading to formation of the blastocyst. Cells of the trophoblast lineage are formed based upon their position in the morula in a complex cascade of inter- and intra-cell signaling events. In primates, implantation of the blastocyst occurs shortly after the blastocyst hatches from the zona pellucida.
The uterus is made receptive to implantation as a result of events controlled largely by production of estrogen and progesterone from the ovaries. During implantation, trophoblasts attach to the receptive uterine epithelium initiating several changes in the endometrium. Vascular changes occur, such as increased permeability of uterine blood vessels, and inflammatory cells are recruited to the implantation site. Proinflammatory cytokines are produced in the uterus and several cellular chances occur. For example, the uterine epithelium is lost and decidual cells undergo an epithelioid transition and proliferate, producing a massively thickened uterine wall. The decidua also contains abundant macrophages, lymphocytes and other bone-marrow derived cells with unusual properties such as reduced alloreactivity, and responsiveness to stimulation by CD3 antibody.
After implantation in humans, distinct populations of differentiated trophoblasts form. Proliferative cytotrophoblast stem cells are anchored to basement membranes surrounding a stromal core in two types of chorionic villi. In floating villi, cytotrophoblast stem cells detach from the underlying basement membrane and fuse to form a syncytium, a polynucleate cell, which covers the villus and is in direct contact with maternal blood. In anchoring villi, cytotrophoblast stem cells differentiate, by detaching from their basement membrane and aggregating to form columns of mononuclear cells which attach to and invade the uterine decidua (interstitial invasion) and its arterial system (endovascular invasion). Interstitial invasion puts cytotrophoblasts in direct contact with the highly specialized subset of leukocytes that are home to the uterus during pregnancy. Endovascular invasion puts cytotrophoblasts, like the syncytiotrophoblasts covering the anchoring villi, in direct contact with maternal blood. Thus, antigen presentation by trophoblasts at the maternal-fetal interface is an important component of maternal immunological responses during pregnancy.
MHC class I molecules and the peptides they present regulate alloreactivity, see for example Sherman, at al. (1993), Annu. Rev. Immunol. 11: 385. Thus, one key to understanding maternal tolerance of the fetal semi-allograft lies in studying trophoblast expression of class I molecules. The molecule HLA-G, which is expressed by placental cells, was cloned in a search for novel class I genes encoded by the human MHC, see for example Geraghty et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 9145. The gene has an intron/exon organization identical to that of the class 1a genes (HLA-A, -B and -C), and the HLA-G protein product has 86% sequence identity to the class I consensus sequence, see for example Parham et al. (1988) Proc. Natl. Acad. Sci U.S.A. 85: 4005. HLA-G has a lower molecular mass (37-39 kDa) then class 1a molecules due to a stop codon in exon 6 that results in the deletion of all but 6 amino acids in the cytoplasmic tail, see for example Shimizu et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 227. With regard to the 5xe2x80x2 flanking region of the gene, the HLA-G promoter has elements (e.g., AP-1, NFkB) similar to sequences found in class 1a genes, but lacks an interferon response element, suggesting novel transcriptional regulatory mechanisms. The primary HLA-G RNA transcript is also differentially spliced; in addition to the full length mRNA, transcripts are produced that lack either exon two, both exons two and three (see for example Ishitani and Geraghty (1992) Proc, Natl, Acad. Sci. U.S.A. 85: 3947), or exon four (see for example Kirszenbaum et al. (1994) Proc. Natl. Acad. Sci; U.S.A. 91:4209). To what extent these alternatively spliced mRNAs are translated is unclear. A soluble form of HLA-G encoded by an mRNA containing intron 4 was described by Fujii et al. (1994) J. Immunol. 153: 5516.
HLA-A, -B and -C are highly polymorphic, but HLA-G appears to exhibit relatively less polymorphism. Immunoprecipitation of HLA-G from 13 individuals and a human choriocarcinoma malignant trophoblast cell line showed identical two-dimensional electrophoretic profiles, suggesting reduced polymorphism at this locus. Genomic and cDNA sequence data also indicate that HLA-G has relatively limited polymorphism. However, there is suggestion that at least in some populations, i.e., African Americans, HLA-G exhibits substantial polymorphism, see for example van der Ven and Ober (1994) J. Immunol. 153: 5628. Whether HLA-G is complexed with endogenous trophoblast peptides and how this repertoire is affected by its degree of polymorphism remains to be determined.
HLA-G is not generally expressed in non-pregnant adults, making it a suitable marker for the diagnosis and monitoring of pregnancy, and for detecting cytotrophoblasts from biological fluids. In addition, HLA-G levels in the maternal blood are indicative of the vigor of cytotrophoblast invasion and the corresponding health of the placental-maternal interface. Prior to the work of Fisher et al., as described in International Publication No. WO 96/31604, suitable antibodies to HLA-G had not been obtained, due to the high similarity of HLA-G to class 1a molecules which are expressed in adults.
One complication of pregnancy in which an abnormal maternal immune response to the fetus has been implicated is pre-eclampsia. This condition is characterized by development of the classic triad of hypertension, edema, and proteinuria, usually in the third trimester of pregnancy, and is usually associated with signs of neurologic hyperirritability, which may eventually result in grand mal seizures. This pregnancy complication remains one of the major causes of maternal and perinatal mortality in both North American and the developing world, see for example Rochat R W, et al. (1988) Maternal mortality in the United States: report from the Maternal Mortality Collaborative, Obstet Gynecol, 72(1):91-7, and AbouZahr C, et al. (1996) Maternal mortality, World Health Stat Q, 49(2):77-87.
Despite extensive research into pre-eclampsia, the underlying cause or causes remain unknown. Evidence accumulated thus far strongly suggests that the causative agent is the placenta, see for example Redman (1991) Placenta, 12(4):301-8.
Pre-eclampsia can occur with hydatidiform mole or choriocarcinoma, see for example Chun D, et al. (1964) J. Obstet. Gynecol. Br. Commonw. 71:180-184, where no fetus is present. The only cure for this widely occurring and life threatening disease is delivery of the placenta. Elucidating the exact role played by the placenta in this disease has been difficult because fundamental aspects of the normal biology of this interesting organ are not well understood. The commonly associated finding of incomplete cytotrophoblast invasion of the spiral arteries suggests impairment of placentation, see for example Lim et al. (1997) Am. J. Pathol., 151(6):1809-18. This lack of trophoblast invasion in pre-eclampsia could be either a primary event or a secondary response to an abnormal immune reaction to the fetus. Several features of this disease suggest that an abnormal response of the mother to the fetus may be the basic defect in this disease, see for example Redman (1991) Semin Perinatol, 15(3):257-62. Pre-eclampsia is most likely to occur in primagravidas, and is usually less severe or absent in subsequent pregnancies, but it may reoccur in the same mother if she changes sexual partners. This suggests that an inappropriate response to some paternally derived antigen, possibly HLA-G, may result in pre-eclampsia.
HLA-G expression has been found to be decreased in the placental bed of women with pre-eclampsia, see for example Hara N, et al (1996) Am J. Reprod. Immunol., 36(6):349-58. However, there is currently no good predictive test for the development of pre-eclampsia, even though shallow invasion in the first trimester sets up conditions for the clinical signs that manifest later in pregnancy, most commonly late in the third trimester.
Although International Publication No. WO 96/31604 (Fisher et al.) teaches the formation of anti-HLA-G monoclonal antibodies 1B8 and 3F6, for use in the detection of HLA-G, there is no suggestion that these antibodies be used in the detection of pre-eclampsia. Both of these antibodies bind to a subsequence in the al domain of HLA-G. Because these antibodies detect the same region of HLA-G, there is no synergy to be derived from their combined use in detecting HLA-G. There is a need for an improved detection method for HLA-G in which more than one antibody is used, and in which the antibodies used do not compete for the same binding region. Further, there is a need for an HLA-G detection method, such as an ELISA which has a high binding selectivity for HLA-G and low binding selectivity for HLA-A2, HLA-B4, HLA-C and WBC.
Conventional methods for diagnosing pre-eclampsia or for detecting antigens in a biological sample require such a sample to be sent away to a laboratory for analysis, and do not allow rapid detection in a clinical setting.
It is object of the present intention to provide an improved test for the measurement of HLA-G in serum levels.
It is another object to provide antigens by generating specific antibodies to both soluble and membrane bound HLA-G.
It is yet a further object of the invention to provide specific antibodies to HLA-G.
It is still a further object of the invention to provide an improved diagnostic test for pre-eclampsia.
Another object of the invention is to provide a miniaturized detection assay, conducive to obtaining rapid results in a clinical setting.
A method is provided for the purification of naturally occurring of HLA-G from human placenta. The method uses any one of the antibodies 3C/G4, 2C/C8, and 4H84.
The invention provides hybridoma 2C/C8, deposited with the International Depositary Authority of Canada (IDAC), Health Canada, 1015 Arlington St., Winnipeg, Manitoba, R3E 3R2 Canada as Accession Number: IDAC 130900-1, deposited on September 13, 2000. This hybridoma produces 2C/C8 antibodies, and is deposited in accordance with all aspects of the Budapest Treaty.
The invention also provides hybridoma 3C/G4, also deposited with the International Depositary Authority of Canada (IDAC). Hybridoma 3C/G4 was afforded Accession Number: IDAC 130900-2, deposited on September 13, 2000. This hybridoma produces 3C/G4 antibodies, and is deposited in accordance with all aspects of the Budapest Treaty.
It has been found that the monoclonal antibody 2C/C8 for HLA-G has substantially the same binding site an the monoclonal antibody 4H84.
The invention provides a method for detecting HLA-G in a biological sample comprising the steps of: (a) depositing a biological sample on a support having an immobilized anti-HLA-G antibody bound thereto, wherein the immobilized anti-HLA-G antibody binds to a first region of HLA-G; (b) contacting the support having the biological sample deposited thereon with an HLA-G label, wherein the label binds to a second region of HLA-G; and (c) detecting the label. In an optional embodiment of the invention, the immobilized anti-HLA-G antibody is 2C/C8, 3C/G4 or 4H84.
Further, the invention relates to a method for identifying an HLA-G indicative condition in a patient comprising the steps of obtaining a biological sample from the patient, followed by the above-noted method for detecting HLA-G.
The invention also provides a method for determining potential for successful implantation of an embryo comprising the steps of obtaining a sample of a fluid medium incubating the embryo followed by the above-noted method for detecting HLA-G.
According to the invention, there is provided a kit comprising: (a) a support having an immobilized anti-HLA-G antibody bound thereto; wherein the immobilized anti-HLA-G antibody binds to a first region of HLA-G; and (b) an HLA-G label which binding to a second region of HLA-G. According to an optional embodiment of the invention, the HLA-G label comprises a mobile anti-HLA-G antibody having a reporter molecule bound thereto. The reporter molecule may be any molecule which is detectable in a quantitative or nearly quantitative manner. For example, a reporter molecule may be a calorimetric agent, a fluorometric agent, a radioisotope, or an enzymatic agents having a detectable end-point.
By the term xe2x80x9cmobilexe2x80x9d as it is used herein with respect to a mobile anti-HLA-G antibody, it is merely indicating that the antibody is not bound to the support, unlike the immobilized antibody, which is bound to the support. Of course, the mobile antibody may be bound to other molecules, such as the reporter molecule. The mobile antibody may be provided in any manner known in ELISA technology, such as in a fluid solution.
When the HLA-G label is a mobile anti-HLA-G antibody having a reporter molecule bound thereto, an optional embodiment of the invention is provided as follows. The immobilized anti-HLA-G antibody may be consisting of 2C/C8, 3C/G4 or 4H84. The mobile anti-HLA-G antibody is then either 2C/C8, 3C/G4, or 4H84, provided that the mobile and immobilized antibodies bind to different regions of HLA-G, referred to herein as a xe2x80x9cfirst regionxe2x80x9d and a xe2x80x9csecond regionxe2x80x9d. In this way, there is no competitive binding between the mobile and immobilized antibodies. Since 2C/C8 and 4H84 bind to the same region of HLA-G, in this embodiment, either the immobilized anti-HLA-G antibody or the mobile anti-HLA-G antibody is 3C/G4. For example, if the immobilized anti-HLA-G antibody is 2C/C8 or 4H84, the mobile anti-HLA-G antibody is 3C/G4. Conversely, if the immobilized anti-HLA-G antibody is 3C/G4, the mobile anti-HLA-G antibody is either 2C/C8 or 4H84.
The method according to the invention may optionally comprise the step of measuring HLA-G by comparing the quantity of label detected in the biological sample with an HLA-G standard. One possible source of the HLA-G standard is that obtained from a human placenta.
The method according to the invention involves a biological sample. Such a sample may be selected from, but is not limited to amniotic fluid, a medium contacting an embryo, a tissue sample, a blood sample, a medium contacting a tissue sample, and a medium contacting a cell, for example when isolated cells are used.
The inventive method may be used to diagnose or detect an HLA-G indicative condition. In this embodiment, a control value for an HLA-G indicative condition can be compared with the quantity of HLA-G found in the sample. Certain conditions may be indicated if HLA-G is low or absent, while others may be indicated by increased levels of HLA-G. One of skill in the art could easily determine the indicative levels useful in diagnosing a condition. Such HLA-G indicative conditions may include, but are not limited to pre-eclampsia, increased risk of pre-eclampsia, adverse fetal outcome, increased risk of adverse fetal outcome, cancer, or increased risk of cancer development.
Hybridomas 2C/C8 and 3C/G4, both deposited at the International Depositary Authority of Canada on Sep. 13, 2000 and having IDAC Accession Numbers IDAC 130900-1 and 130900-2 are encompassed by the invention. Antibodies produced by these hybridomas, ELISAs involving these antibodies also fall within the scope of the invention.
The method according to an embodiment of the invention may comprise a miniaturized assay conducted on a chip. A chip may refer to a support formed of any plastic or inert polymer such as polystyrene, a cellulose or nitrocellulose support, a silicon-based or metal-based support, or any other platform as is known in the art. According to one embodiment of the invention, an immobilized anti-HLA-G antibody is bound to a polystyrene chip, sized to receive a sample volume ranging from about 5 to 50 xcexcl. A miniaturized assay can be conducted in a clinical setting, for example for determining HLA-G in a sample of embryo culture medium, for predicting successful embryo implantation. Larger or smaller sample volume assays are also within the scope of the invention, but this embodiment is conducive to an on-site test which does not require a sample to be sent away for laboratory analysis. Use of such a chip, for example within a kit would be advantageous for determining potential for successful implantation of an embryo in in vitro fertility procedures.
The invention further provides a method for selecting an embryo for in vitro fertilization (IVF) comprising the steps of detecting soluble HLA-G secreted by an embryo into an incubation medium, and choosing an embryo secreting soluble HLA-G at a minimum level of 0.01 xcexcg/ml of incubation medium. Optionally, this method may be combined with the evaluation of embryo cleavage rate, and based on both parameters, an embryo is chosen for IVF. In this method for selecting an embryo, the step of detecting soluble HLA-G may be conducted according to the method for detecting HLA-G according to the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described.
The term xe2x80x9cantibodyxe2x80x9d refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin, antibody, structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about, for example, 100 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies can exist, for example, as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)xe2x80x22 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab with part of the hinge region broken. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fabxe2x80x2 fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
The term xe2x80x9cbiological sample or fluidxe2x80x9d refers to material derived from a living organism, including but not limited to blood and blood derivatives, cervicovaginal secretions, amniotic fluid, cord blood, urine, tissues, bones and cells.
The term xe2x80x9cblood samplexe2x80x9d as used herein includes whole blood or derivatives of whole blood well known to those of skill in the art. Thus a blood sample includes the various fractionated forms of blood such as plasma or serum and whole or fractionated blood which additionally comprises various diluents as may be added to facilitate storage or processing in a particular assay. Such diluents are well known to those of skill in the art and include various buffers, anticoagulants, preservatives and the like.
The term xe2x80x9cHLA-Gxe2x80x9d refers to human leukocyte antigen G and unless otherwise stated includes both the soluble and insoluble forms. The term may in appropriate context refer to either the antigen or the genetic locus.
The term xe2x80x9cimmunoassayxe2x80x9d is an analysis or methodology that utilizes an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of at least one particular antibody to isolate, target, or quantify the analyte.
The terms xe2x80x9cisolatedxe2x80x9d, xe2x80x9cpurifiedxe2x80x9d, or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
The term xe2x80x9cnucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotide that can function in a similar manner as naturally occurring nucleotides, for example, incorporating conservative substitutions as is well known in the art.
The term xe2x80x9cnucleic acid probexe2x80x9d refers to a molecule which binds to a specific sequence or subsequence of a nucleic acid. A probe is preferably a nucleic acid which binds through complementary base pairing to the full sequence or to a subsequence of a target nucleic acid. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
A xe2x80x9clabelxe2x80x9d is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, calorimetric, enzymes, for example, as commonly used in ELISA, biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available can be made detectable.
A xe2x80x9clabeled nucleic acid probexe2x80x9d is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
The term xe2x80x9crecombinantxe2x80x9d when used with reference to a cell indicates that the cell contains nucleic acid with an origin exogenous to the cell. Thus, for example, recombinant cells replicate and/or express genes that are not found within the native (non-recombinant) form of the cell.
The term xe2x80x9cidenticalxe2x80x9d in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
The phrases xe2x80x9cspecifically binds toxe2x80x9d, xe2x80x9cspecifically hybridizes toxe2x80x9d or xe2x80x9cspecifically immunoreactive withxe2x80x9d, when referring to an antibody indicate a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological substances. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in a biological sample or fluid. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity of binding with a particular protein.
Herein, a particular antibody and the cell which produces the antibody may be referred to by the same term, however, one of skill in the art can easily distinguish whether it is the antibody or the cell to which any particular instance pertains. Thus, a hybridoma cell line may be referred to by the name of its defining antibody.